Although many vectors exist for Escherichia coli and closely related species, there are few broad host range vectors that can be conjugated into a large variety of Gram-negative bacteria. We have constructed a broad host range vector, pMJ445, that facilitates the analysis of divergent promoters in Gram-negative bacteria. The vector was validated using two intergenic regions derived from gene clusters involved in hydantoin hydrolysis, from the environmental isolates Pseudomonas putida and Agrobacterium tumefaciens. The DNA sequences analysed were capable of activating expression of the reporter enzymes, β-glucuronidase and β-galactosidase, present on pMJ445, indicating the presence of divergent promoters in the sequences selected. In addition, we demonstrated that pMJ445 can be applied to gene regulation studies.